Multiple antigen peptide system having adjuvant properties, vaccines prepared therefrom and methods of use thereof

ABSTRACT

A multiple antigenic peptide system is disclosed that comprises a dendritic core and peptide and a lipophilic anchoring moiety. This particular combination has as an advantage that it eliminates the need for the inclusion of adjuvants found to be toxic to humans, and facilitates the exponential amplification of the antigenic potential of a vaccine prepared therefrom, as noncovalent amplification by a liposome or micellar form is possible. Further, multiple different antigenic peptides may be attached so that the system may be prepared for administration to concurrently treat diverse ailments, such as for example, AIDS and influenza. The present multiple antigen peptide system is capable of eliciting an immune response when injected into a mammal, and accordingly, vaccines prepared from the system and methods of use including therapeutic protocols are included.

The present invention was made with partial assistance from Grant No.A128701. The Government may have certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.07/877,613, filed May 1, 1992, which is now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of immunology, andparticularly to the preparation and administration of vaccines forprevention and treatment disease states such as HIV infection.

Highly specific and immunogenic antigens are preferred as vaccines.While the immunogenicity of an antigen can be increased by coupling aprotein carrier to the antigen, this approach has several drawbacks.First, if the carrier is large, significant humoral immune response canbe directed against the carrier rather than the antigen. Second, a largecarrier can suppress humoral response to the antigen. Finally, thecoupling of an antigen to a protein carrier can alter the immunogenicdeterminants of the antigen.

Multiple antigen peptide systems (MAPS) are designed to overcome theproblems observed with conventional protein careers. Most MAPS arecomposed of several peptide antigens covalently linked to a branching,dendritic core composed of bifunctional units (e.g., lysines). Thus, acluster of antigenic epitopes form the surface of a MAPS and a smallmatrix forms its core. As a result, the core is not immunogenic. MAPShave been used to prepare experimental vaccines against hepatitis (Tamet at., Proc. Natl. Acad. Sci. USA 86: 9084, 1989), malaria (Tam et at.,J. Exp. Med. 171: 299, 1990), and foot-and-mouth disease. A furtheradvantage of MAPS is that they are chemically unambiguous. This allowsdifferent epitopes, such as B cell and T cell epitopes, to be arrangedin a particular arrangement and stoichiometry.

Specific MAPS have been and are in development for use in immunizationagainst HIV. For example, European Patent Publication No. 0 339 695published 11 Feb. 1989 describes a process for preparing MAPS byreacting a branched structure based on an amino acid such as lysine orornithine with a separately synthesized antigenic compound.

European Patent Publication No. 0 328 403, published 16 Aug. 1989describes particular peptides that are specifically immunoreactive withantibodies to HIV and suggests that MAPS which include these peptidescan be used for immunization to prevent HIV infection.

Hart et al. (J. Immunol., 145: 2677, 1990) report that a syntheticpeptide construct which includes amino acids 428-443 and 303-321 ofHIV-I-III, envelope protein gp120, when used as a carrier-free immunogenin primates, can induce a high titer of neutralizing anti-HIV antibodiesand can induce T cell proliferative response against native HIV-1 gp120.

Palker et al. (Immunology 142: 3612, 1989) describes the use of a 16amino acid T cell epitope from HIV-1-IIIB fused to a synthetic peptidewhich includes a type-specific neutralizing determinant of a particularHIV-1 strain (III. 1 MN or RF) to immunize goats. Both T cells and Bcells responded to epitopes within the type-specific neutralizingdeterminant.

PCT Application Publication No. WO 90/11778 published 18 Oct. 1990discloses multiple antigen peptide systems in which a large number ofeach of T cell and B cell malarial antigens are bound to the functionalgroups of a dendritic core molecule.

In Copending U.S. application Ser. No. 07/744,281, now abandoned, by Tamet al., a particular multiple antigen peptide is prepared for use as avaccine for the treatment of HIV infection that incorporates particularpeptides derived from the HIV-1 III_(B) envelope protein as well as theV3 loop of the gp120 protein of HIV 1-MN. This peptide systemdemonstrates the capability of generating a humoral response and thedevelopment of antibodies, and seeks to elicit a T cell response by theinclusion of a T cell epitope. The in vivo administration of thispeptide requires the inclusion of an adjuvant as a means of enhancingthe humoral response.

More generally, most vaccine strategy developed today particularlyagainst human immunodeficiency virus (HIV) infection has been directedtoward the humoral response of generating neutralizing antibodies.Recent advances in mapping antigens involved in immune responses haveallowed detailed characterization of epitopes that confer neutralizing,T-helper and T-cytotoxic responses. These developments have led toconsideration of including the T-cytotoxic response along with humoralimmunity in the design of peptide-based vaccines.

As noted above and elsewhere, traditional methods for preparing peptidevaccines that present peptides as macromolecules through conjugation toprotein carriers or polymerization are often unable to induce cytotoxicT lymphocytes (CTL) response in vivo. Use of an adjuvant in theimmunizing protocol has the advantage of enhancing the humoral responsebut has mixed results in priming specific CTL response. Furthermore, themost popular adjuvant used in laboratory animals, such as Freund'scomplete adjuvant, is too toxic and unacceptable for humans. Ideally,protection against viral infection is best provided by both humoral andcell-mediated immunities, including long-term memory and cytotoxic Tcells.

Specifically, the human immunodeficiency virus (HIV), the etiologicagent of the acquired immunodeficiency syndrom (AIDS), has become animportant objective for various vaccine developments. The predominantvaccine strategy has focused on the use of the envelope protein antigensgp120 and gp160 of HIV-1 produced by the recombinant DNA technology.However, the full promise of their use in vaccines will not be realizedunless they are administered along with an effective adjuvant.

An adjuvant is usually a non-toxic agent that provokes specificresponses to antigens. There is a wide spectrum of mechanisms by whichan adjuvant functions. It can function by creating a depot at the siteof injection that prolongs the release of antigens withantigen-presenting cells. It may also function by activating macrophagesto release cytokines and mediators which in turn activate effector Tcells or antibody-forming B cells. The net result is that an adjuvantaugments specific humoral and cell-mediated immunities with a lower doseof antigen required.

Many seemingly unrelated agents have been used as adjuvants and thecommonly used adjuvants can be broadly categorized into four groups. Thefirst, and the only clinically acceptable group, belongs to the gels ofaluminium (e.g. alum) and calcium salts. However, alum is a weakadjuvant and its formulation in laboratory tests of HIV and SIV antigenshas been found to be inadequate. The second, and perhaps the most potentgroup, includes pure compounds and undefined mixtures derived frommycobacterial cell walls. Mixtures such as Freund's complete adjuvant(FCA) and lipopolysacchaxides (LPS) are the best known examples.However, FCA and LPS produce side effects. They are pyrogenic and inducearthritis in rats and anterior uveitis in rabbits.

A need therefore exists for the development of a vaccine formulationthat offers improved immunity in both categories while avoiding thedrawbacks of traditional adjuvant materials.

SUMMARY OF THE INVENTION

In accordance with the invention, a multiple antigenic peptide system isdisclosed that comprises a dendritic core and at least one peptide and alipophilic membrane anchoring moiety attached thereto, wherein the themultiple antigen peptide system, when injected into a mammal, is capableof eliciting a full immune response including both humoral and cytotoxicT cell responses.

More particularly, a synthetic peptide-based vaccine may be preparedfrom the present peptide antigen system, that is effective in providingboth humoral and cell-mediated immunities, and is safe in its exclusionof Freund's complete adjuvant. The present invention employs amacromolecular assemblage principle that allows amplification of peptideantigens to a macromolecule. The resulting macromolecule bears theimmunological mimicry of the external-surface coat protein of apathogen.

The lipophilic anchoring moiety useful in the present invention maycomprise a lipoamino acid, liposomes, saponin derivatives alone or inadmixture with cholesterol, and suitable surfactant materials such asthe PLURONICS comprising a mixture of long chain polyoxyethylenes andpolyoxypropylenes. Particularly, a long side chain bearing amino acidsuch as tripalmitoyl-S-glyceryl cysteine (P3C) has been determined to besuitable. Analogs of P3C such as P2C and materials prepared from serineand lysine, the latter exemplified by palmitoyl lysine, as well ascysteine are likewise included herein.

The antigen system of the present invention is versatile, as theexponential magnification possible by the preparation of multipleantigens facilitates the presentation of multiple and different suchantigens, so that immunization for several different and distinctinfective stimuli is possible. For example, a single vaccine prepared inaccordance herewith may present antigens for HIV, influenza and malaria,by the attachment to the core of coat proteins for each of thesestimuli.

In a preferred embodiment, the multiple antigenic peptide systemincludes a T cell epitope. The T cell epitope may be covalently linkedin tandem to the peptide. By "T cell epitope" is meant a peptide capableof eliciting a proliferative T cell response. Preferably, the T cellepitope is at least seven amino acids long.

In related aspect, the invention features a method for eliciting animmune response against HIV in a mammal. The method includesadministering to the mammal the multiple antigen peptide systemdescribed above.

The invention further relates to a vaccine which includes animmunologically effective amount of the multiple antigen peptide systemdescribed above. The invention also extends to a method for generatingantibodies by admininstering to a mammal an antibody-generating amountof the multiple antigen peptide system of the invention.

The scope and content of the invention will be better understood from areview of the detailed description which proceeds with reference to thefollowing illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of MAP-P3C.

FIG. 2 is a graph of the mitogenic activity of the B1M-P3C. BALB/c mousespleen cells (3.3×10⁶ cells/ml) were grown for 72 hr in the presence ofB1M-P3C (▪) or B1M (□). 24 hr before harvesting, cells were pulsed with1 μCi [³ H]thymidine.

FIG. 3 is a graph showing IL-2 production of lymphocytes primed withB1M-P3C. Splenocytes from mice immunized with 50 μg of B1M-P3C werecultured in presence of B1 (▪) or a control peptide from the malariacircumsporozoite protein [(Asn-Ala-Asn-Pro)₃ MAP] (□).

FIG. 4 is a graph showing induction of HIV-1 envelope-specific CTL.Spleen cells from mice immunized with B1M-P3C in liposome (A) or free(B) were restimulated with B1. Cytotoxic activity was tested on P815target cells alone (◯), presensitized with a control peptide T1 derivedfrom the C4 of gp120, aa 429-443 (Δ), presensitized with B1(▪), infectedwith vaccinia virus WR (□) or with vaccinia virus v-env5 expressinggp160 (▴).

FIG. 5 is a graph showing IgG subclass responses to i.p. inoculatedB1M-P3C/liposome. Plates coated with B1 antigen and affinity purifiedalkaline phosphatase-conjugated antibodies directed against specific IgGsubclasses were used.

FIG. 6 is a graph showing the cytotoxic response of T-cells derived frommice immunized with B1M-P3C/liposome. The cells were taken 5 monthsafter the last boost and restimulated in vitro with the V3 peptide. CTLactivity was assayed on untreated, or recombinant vaccinia virus(v-env5, containing the HIV-1 envelope gene of the BRU isolate) infectedtarget cells (P815), or cells preincubated with B1 peptide (0.8 μM).

FIG. 7 depicts the schematic structure of a lipoMAP in a lipid matrix.

FIG. 8 depicts the schematic representation of tripalmitoyl cysteineconjugate of MAP bearing B2 peptides (B2M-P3C) with a disulfide linkage.

FIG. 9 schematically depicts structural formulas illustrating (A)β-alanyl-lysine; (B) Asymmetrical MAP core (M) and (C) symmetrical MAPcore (SM).

FIG. 10 comprises a schematic representation of MAP conjugatescontaining (A) palmitoyl groups linked to the side chain of serines(B2SM-PS3); (B) L-Ser-D-Ser linker (B2SM-D-PL3); (C) zero to fourpalmitoyl lysines.

FIG. 11 comprises graphs showing the immunogenicity of MAP-PLs, varyingthe linker and of the lipophilic moiety. Antibody response induced inCD-1 mice by the immunization with B2SM-PL3 (), B2SM-D-PL3, (o),B2SM-PS3 (▴), as measured by ELISA.

FIG. 12 shows the influence of increasing numbers of palmitoyl sidechain on the immunogenicity of B2SM-PLs. The antibody response wasmeasured by ELISA in the sera of mice immunized with B2SM-PL2/liposomes(), -PS3/liposome (▴), -PLA/liposomes (▪) or with the free constructsB2SM (x). B2SM-PL1 (□), -PL2 (o), -PL3 (Δ), -PL4 (□). (A) antibodyresponse against B2 peptide; (B) against gp120.

FIG. 13 comprises graphs illustrating the induction of anti-HIV CTL.Cytotoxic activity in the spleen cells of BALB/c mice immunized withB2SM-PL2 free (A) or B2SM-PL2/liposome (B) was assayed on syngeneic P815target cells coated with the antigenic peptide (closed symbols) or anuntreated P815 cells (open symbols).

FIG. 14 comprises a structural depiction of the preparation oftripalmitoyl cysteine conjugate of MAP bearing B2 peptides (B2M-P3C)with a disulfide linkage.

DETAILED DESCRIPTION

In its broadest aspect, the invention relates to multiple antigenpeptide systems that include a lipophilic membrane anchoring moiety thatconfers adjuvant properties among its advantages. The dendritic core andthe multiplicity of antigens attached thereto, however, are thecharacteristics of the antigenic materials that the present inventor andothers have already developed. Accordingly, the following discussion isby way of background to the extent that the MAPS portion of the presentsystem shares its origins with such previously prepared materials.

Multiple antigen peptide system (MAPS) is the commonly used name for acombination antigen/antigen carrier that is composed of two or more,usually identical, antigenic molecules covalently attached to adendritic core which is composed of bifunctional units. The dendriticcore of a multiple antigen peptide system can be composed of lysinemolecules. For example, a lysine is attached via peptide bonds througheach of its amino groups to two additional lysines. This secondgeneration molecule has four free amino groups each of which can becovalently linked to an additional lysine to form a third generationmolecule with eight free amino groups. A peptide may be attached to eachof these free groups to form an octavalent multiple peptide antigen.Alternatively, the second generation molecule having four free aminogroups can be used to form a tetravalent MAPS, i.e., a MAPS having fourpeptides covalently linked to the core. Many other molecules, includingaspartic acid and glutamic acid, can be used to form the dendritic coreof a multiple peptide antigen system. The dendritic core, and the entireMAPS may be conveniently synthesized on a solid resin using the classicMerrifield synthesis procedure.

Multiple antigen peptide systems have many advantages as antigen carriersystems. Their exact structure and composition is known; the ratio ofantigen to carrier is quite high; and several different antigens, e.g.,a B cell epitope and a T cell epitope, may be attached to a singledendritic core. When both a B cell epitope and a T cell epitope arepresent it is preferable that they are linked in tandem on the samefunctional group of the dendritic core. Alternatively the T cell epitopeand the B cell epitope may be on separate branches of the dendriticcore. Preferably, the T cell epitope is a helper T cell epitope; howevera cytotoxic T cell epitope may also be used.

Useful T cell epitopes may be derived from the HIV-1 envelope protein.However, it is not necessary that the B cell epitope and the T cellepitope both be derived from the HIV-I gp120 envelope protein. T cellepitopes from different HIV-I proteins (e.g., those encoded by the nef,gag, tat, roy, vif, pol, vpr, vpu, or vpx genes), different retrovirus,or unrelated organisms (e.g., malarial antigens or tetanus toxoid) maybe used. T cell epitopes can be identified by a T cell proliferationassay.

Multiple antigen peptide systems and methods for their preparation aredescribed more fully in PCT Publication No. WO 90/11778 and EuropeanPatent Publication No. 0 339 695, both of which are hereby incorporatedby reference.

As stated earlier, the present invention extends to the preparation ofthe multiple antigen peptide system herein including the internaladjuvant quality imparted by the macromolecular assemblage approach. Thesystem so prepared may be formulated as a vaccine having a variety ofadvantages among them the ability to be adapted for both parenteral andoral administration. Accordingly, the invention extends to thepreparation of vaccines and their administration to prevent thedevelopment of viral infections, and to elicit an immune response,and/or to raise antibodies to such pathogens.

As stated earlier, the invention also extends to the elicitation of animmune response in a host by the administration of compositionsincluding the present multiple antigen peptide system, as well as theimmunization of a host by the administration of a vaccine prepared inaccordance herewith. The present multiple antigen peptide system mayfurther be prepared with a variety of vehicles including encapsulationwithin liposomes, for greater efficiency of delivery and concomitantlyreduced dosage. The preparation of liposomes is well known in the art.

The amplification that is characteristic of the multiple antigen peptidesystem of the present invention is made possible by two syntheticcomponents (FIG. 1), (i) A scaffolding consisting of an oligomericbranching lysine is used to amplify the peptide antigen 4-fold to give amultimeric structure. This covalent amplification, known as multipleantigen peptide system (MAPS), has been effective in inducing strongimmune responses (12,13). (ii) A lipophilic membrane-anchoring moiety atthe carboxyl terminus of MAPS enables further noncovalent amplificationby a liposome or micellar form. Such a macromolecular assemblage willamplify the MAP antigens many fold.

Several lipophilic moieties have been studied and are presented herein.The first mentioned and the one that has proved particularly successfulis the tripalmitoyl-S-glyceryl cysteine (P3C). P3C, which is a lipoaminoacid from Escherichia coli, is a B cell mitogen, a nontoxic adjuvant,and can induce CTL in vivo when covalently linked to a peptide antigen(14).

In addition and as illustrated in Example 3 later on herein, palmitoyllysine has also been prepared and tested with efficacy, and theinvention accordingly extends to the use of this lipophilic structure aswell.

The antigen for the presently illustrated model is located in the thirdvariable domain (V3 loop) of gp120, the envelope glycoprotein of HIV-I,which is the principal target for vaccine development against AIDS (4).The V3 loop of IIIB strain, amino acid sequence 291-343, contains aninvariant disulfide bridge and a type-II β turn with the sequenceGly-Pro-Gly at its crest. Antibodies raised to the V3 loop neutralizedthe in vitro infectivity of HIV, and the principal neutralizingdeterminant has been found to be centered at the β turn. Our previousstudies in mice (15) have found that a 24-residue peptide of the V3loop, referred to as B1 (sequence 308-331) and SEQ ID NO: 12 inco-pending application Ser. No. 07/744,281, abandoned in favor ofcontinuation application Ser. No. 08/120,310, filed Sep. 13, 1993, nowabandoned, contains the minimal sequence that consists of neutralizingand T-helper epitopes. In addition, this B1 sequence also contains aT-cytotoxic epitope (sequence 315-329) (6). As shown herein, a vaccinemodel using the peptide B1 by the macromolecular assemblage approach(referred to as B1 in a tetravalent MAPS (B1M format linked to P3C(B1M-P3C) induces specific antibodies against gp120 that neutralizevirus infectivity in vitro and elicits CTL in vivo.

The following examples illustrate the preparation of vaccines inaccordance with the invention, and further, present data confirmingtheir utility.

EXAMPLE 1

In the following experiments the preparation and comparative testing ofa model vaccine based on the present invention is set forth. As a reviewof the following data will reveal, the vaccine exhibited both improvedhumoral and cytotoxic activity.

Materials and Methods

Synthesis of B1M-P3C. Synthesis was accomplished manually by a step-wisesolid-phase procedure (16) on 9-flourenylmethoxycarbonyl (Fmoc)-Ala-OCH₂-resin (17) (0.3 mmol/g of resin). After removal of the Fmoc group by20% piperidine in dimethylformamide, the Ala-resin was coupled to apremade unit of Fmoc-Lys (P3C) (1.1 molar equivalent) viadicyclohexylcarbodiimide/hydroxybenzotrizole in CH₂ Cl₂. The P3C wasprepared as described (14, 18) and contained the configuration ofN-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-(propyl)]-(R)-cysteine. Twoconsecutive serines as a linker were coupled to the Lys(P3C) beforeadding the two levels of Fmoc-Lys(Fmoc) to give a tetrabranching[Fmoc-Lys(Fmoc)]₂ -Lys-Ser-Ser-Lys(P3C)-Ala resin. The protecting groupscheme for the synthesis was as follows: Fmoc for the N.sup.α aminogroup and the tertbutyl side chain protecting groups for thetrifunctional amino except Arg(Pmc) and Asn(Trt). Three equivalents ofFmoc-amino acid were used for each coupling by the DCC/HOBt method.Coupling was monitored by the qualitative ninhydrin test (19).Deprotection by 20% piperidine in DMF (10 min) was preceded by a 2-minwash. The completed peptide was cleaved from the resin by CF₃ CO₂ H/2%dimethylsulfide/2# anisole/1% ethanedithiol. The MAPS peptides werepurified by precipitations in CF₃ CO₂ H/tertbutyl methyl ether.Similarly, synthesis of B1 was accomplished by the Fmoc/tertbutylstrategy and the peptide was purified by reverse-phase HPLC. Allpeptides gave satisfactory amino acid analyses.

Preparation of positively charged liposome

Liposomes were prepared as described by Gregoriadis et al. (20).Briefly, 56 mg of egg lecithin, 8.4 mg of cholesterol and 1.8 mg ofstearylamine were solubilized in CHCl₃ in a 100 ml round bottom flask.P3C (0.24 mg) was added to liposomes made with B1M. The organic solventwas removed under vacuum using a rotary evaporator to form a thin filmof lipid on the wall of the flask. After drying, nitrogen was keptflushing in the flask for 10 min. Two millileters of 10 mM at pH 7.4,containing 2.5 mg of peptide, was added into the flask. Shaken manuallyfor 10 min, the suspension was then allowed to stand at room temperaturefor 2 hr. The resulting milky solution was sonicated 45 min (LaboratorySupply, Indianapolis) until the solution became opalescent. Aftersonication, free B1M-P3C was separated from the liposomes on Sepharose6B; liposomes were then filtered on 0.45 μm filter and kept undernitrogen.

Immunization procedure and ELISA.

Dunkin-Hartley guinea pigs (3 per group) were immunized subcutaneouslywith 100 μg of peptide on day 0 and 14, and with 50 μg on days 30 and45. They were bled two weeks after the last boosting. Control guineapigs were immunized with the same protocol using a noncovalent mixtureof B1M, P3C, and liposome. BALB/c mice (5 per group), 6 to 8 weeks old,were immunized intraperitoneally four times with 1 to 100 μg of B1M-P3Cat two to three weeks intervals and bled two weeks after the lastboosting. Control mice were immunized with a noncovalent mixture of 50μg of B1M, P3C and liposome. Antisera were used without purification.ELISA was used to test antisera for their ability to react with B1 (5 μgper well) or recombinant gp120, IIIB isolate (0.1 μg per well)(Repligen, Cambridge, Mass.).

Functional assays.

Fusion inhibition assay was performed on CD4 positive cells CEM-T4(ATCC) which were infected with either wild type WR isolate orrecombinant vaccinia virus (vPE16 recombinant vaccinia vector expressingthe HIV-1 envelope glycoprotein gp160 of the IIIB isolate provided byDr. B. Moss, obtained through the AIDS Research and Reference ReagentProgram, division of AIDS, NIAID) at multiplicity of infection of 10.Antisera were added to the cultures 1 hr post infection and syncytiawere counted 24 hr post infection (15). For CTL assay,

BALB/c mice were immunized with 100 μg of antigen. Three to eight weekslater, immunized spleen cells (2.5×10⁶ per ml) in RPMI 1640 medium/10%fetal calf serum/2 mM glutamine/50 μm2-mercaptoethanol/antibiotics(GIBCO), complete culture medium were restimulated for 6 days in vitrowith 0.4 μM of peptide B1 in 24-well culture plates.

The cytolytic activity of the restimulated cells was tested using a 4 hrassay with ⁵¹ Cr-labeled syngeneic cells P815 (H-2^(d)). The targetcells were infected with vaccinia viruses v-env5 (recombinant vacciniavirus expressing the complete envelope gene of HIV-1 was provided by Dr.S.-L. Hu) at multiplicity of infection of 50, or pulsed with syntheticpeptide (0.8 μM for 2 hr at 37° C.) prior to labeling. The % specific ⁵¹Cr release was calculated as 100×[(experimental release--spontaneousrelease)/(maximum release--spontaneous release)]. Maximum release wasdetermined from supernatants of P815 cells lysed by the addition of 5%Triton X-100 and spontaneous release from target cells incubated alone.SEM of triplicate cultures were all <5% of the mean. Neutralizationassay by inhibition of the reverse transcriptase activity was performedas reported by Ho et at. (21 ) on HIV-infected H9 cells.

IL-2 production and B-cell proliferation.

Splenocytes of mice primed intraperitoneally 10 days before with 50 μgof B1M-P3C were dispensed in 24 well-plates at the concentration of4×10⁶ cells per well in complete culture medium. Peptides at variousconcentrations were added to the cell suspension. Medium alone was addedto other wells to assess the IL-2 production in absence of antigenicrestimulation. After 36 hr incubation at 37° C., supernatants werecollected, centrifuged, and added to IL-2 dependent cytotoxic T-lymphoidline (CTLL-2) to determine their IL-2 activity.

Briefly, 50 μl of supernatant was added to 50 μl of CTLL-2 suspension(8×10⁴ cells per ml) in a 96-well microtiter plate. Cells were harvestedafter one day incubation. During the last 5 hr of incubation, each wellwas pulsed with 1 μCi of [³ H]thymidine (1 Cl=37 (3 Bq). Results wereexpressed in units of IL-2 per ml (means of triplicates) for each group.B-cell proliferation assay was performed on spleen lymphocytes grown for72 hr in RPMI 1640 medium/3.3% fetal calf serumglutamine/2-mercaptoethanol/antibiotics at a cell density of 3.3×10⁶cells per ml in flat-bottom microtiter plates (0.2 ml per well).Antigens were added at the beginning of the culture. Before harvesting,cultures were pulsed for 24 hr by adding 1 μCi of [³ H] thyroidinc toeach well. The results are as reported as means of triplicatedeterminations (standard errors were <10% of the mean) of arepresentative experiment.

RESULTS

Synthesis of B1M-P3C.

B1M-P3C (FIG. 1) was synthesized in two parts. (i) P3C was achieved in asolution synthesis in six steps according to Wiesmm uller et al. (18)and linked to the side chain ε-amino group of Fmoc-Lys-phenacyl ester.The phenacyl ester protecting group was subsequently removed to give anisopeptide, Fmoc-Lys (P3C). (ii) The synthesis of B1M which containedthe B1 antigen and the lysine core matrix was achieved by thesolid-phase method (13,16) with Fmoc-Ala-OCH₂ -resin (17). Fmoc-Lys(P3C)as a premade unit was first attached to the Ala-OCH₂ -resin, followed bya diserine spacer prior to the synthesis of a trilysine core matrix ofMAPS and the B1 sequence. The design of linking P3C to the side chain ofthe lysine spacer (Ser-Ser-Lys) at the carboxyl terminus of the MAPS wasintended to provide flexibility for the P3C to serve as alipid-anchoring moiety without interfering with the antigen organizationat the amino terminus. Because the secondary ester bond in P3C waslabile to HF, the solid-phase synthesis was performed with the Fmocchemistry (22) in combination with the Wang resin (17) so that the finalcleavage could be carried out in a mild acid such as CF₃ CO₂ H. Thesynthesis, performed manually, was rigorously monitored for thecompletion of each coupling step (22) to avoid deletion peptides.B1M-P3C was obtained after TFA cleavage from the resin support and waspurified by repeated precipitations.

The advantage of this direct approach was its simplicity. Otherunambiguous routes for the preparation of B1M-P3C by the segmentapproach have also been developed (23). P3C in B1M-P3C allowed selfaggregation in aqueous solution and efficient incorporation inliposomes. About 80% of B1M-P3C was found to be associated withliposomes while only 2% of B1M without P3C was found to be entrapped byliposomes. Both preparations of B1M-P3C in aggregated form in solution(B1M-P3C/free) or associated with positively-charged liposomes(B1M-P3C/liposome) were tested in animals for humoral and CTL responses.

B-cell mitogenic activity and humoral response of B1M-P3C.

Mouse spleen cells were used to demonstrate that the mitogenic activityof the P3C was retained in B1M-P3C (FIG. 2). The mitogenicity wasdose-dependent with increased incorporation of [³ H]-thymidine in spleencells with escalated concentrations of B1M-P3C. B1M without P3C did notshow any mitogenic activity.

The ability of B1M-P3C/liposome or free, without any adjuvant, to inducehumoral response was studied in mice and guinea pigs. High-titeredantibodies were found in sera from animals immunized four times withboth preparations and treated with B1 (linear peptide 308-331), B1M, orgp120 in a ELISA assay, the data from which is set forth in Table 1,below.

                  TABLE 1                                                         ______________________________________                                        Immune Response to B1M - P3C                                                         Antibody Titer                                                                (× 10.sup.-3).sup.A                                                                  Inhibition                                                                            Syncytium                                                                              RT                                       Immunogen                                                                              Peptide  gp120     formation.sup.b                                                                        activity.sup.c                           ______________________________________                                        Mouse                                                                         Control  <0.01    <0.01     0        ND                                       Liposome 10       4         20       8                                        Free     4.3      2         10       8                                        Guinea Pig                                                                    Control  6.8      1.2       0        0                                        Liposome 6.5      3.2       20       8                                        Free     8.2      3.3       10       8                                        ______________________________________                                         RT, reverse transcriptase; ND, not done.                                      .sup.a ELISA titers represent the reciprocal of the endpoint dilution         (serum dilution at which OD is 0.2 unit). Mice were immunized with 100        μg of B1M - P3C, and control group were immunized with 50 μg of a       noncovalent mixture of B1M/P3C/liposome.                                      .sup.b Fusion inhibition titers are dilutions reducing the number of          syncytial foci by 90%.                                                        .sup.c Neutralization titers are the reciprocal of a dilution reducing RT     activity by 87%.                                                         

There was no significant difference between B1M-P3C/free orB1M-P3C/liposome, but there was a dose-response of titers withincreasing immunizing doses. Tilers from mice immunized with 100 μg ofB1M-P3C/liposome were 2- and 4 fold higher than those immunized with 50or 10 μg (data not shown). Both preparations of B1M-P3C elicitedantibodies in mice and guinea pigs that showed ability to neutralize HIVas shown by the inhibition of syncytia formation in vPE16-infected cellsand the reverse transcriptase activity of HIV IIIB-infected H9 cells(Table 1 ). Control mice immunized with a noncovalent mixture ofB1M/P3C/liposome did not develop detectable antibodies against B1 orgp120. In contrast, this noncovalent mixture elicited significant titersin guinea pigs. However, both sera of control mice and guinea pigs hadno effect on the ability to inhibit the syncytia formation or thereverse transcriptase activity.

Cytokine production and CTL response.

B1M-P3C induced CD4⁺ T-helper cell response in immunized mice (FIG. 3).IL-2 activity was found in the supernatant of spleen cells restimulatedwith B1. A control and unrelated peptide from the CS protein[(Asn-Ala-Asn-Pro)₃ MAP)] did not show any activity. Spleen cells ofmice immunized with B1M-P3C free or in liposome were assayed for theirability to lyse target cells preincubated with B1 or infected withvaccinia virus expressing gp160. As shown in FIG. 4, B1M-P3C, inliposome or free, elicited CD8+CTL in mice against vaccinia virusinfected cells (v-env5) and B1 peptide-coated cells. The CTL responsewas mediated by CD8+lymphocytes (unpublished work).

EXAMPLE 2

In the following experiments, further vaccines were prepared and testedboth as to their preparation and as to their administration. The resultsare further confirmatory of the effectiveness and particularly of theadjuvant capability that the present system manifests.

As discussed herein, the vaccine model (B1M-P3C) is designed to mimicthe glycoprotein gp120 anchored on the virus external membrane. Itcontains four components (starting from the amino terminal of theconstruct): four identical peptide antigens (aa 308-331), an oligolysinescaffolding (Lsy₂ Lys), a spacer (Ser-Ser-Lys) and a hydrophobic moiety(P3C) attached to the Lys side chain of the tripeptide spacer. Becauseof the hydrophobicity of P3C, B1M-P3C is able to aggregate in aqueoussolution or anchored in liposomes to form a large macromolecularassemblage. Both preparations were tested in mice to show their efficacyin the induction of antibodies and CTL response.

Materials and Methods

B1M peptide was synthesized by stepwise solid phase synthesis onFmoc-Ala-Wang resin. It was covalently linked to the premade P3C using 2serine residues as a spacer (30). Liposomes were made following themethod of Gregoriadis (20). BALB/c mice (5 each group) were immunizedi.p. or s.c. with 50 μg of B1M-P3C free or in liposomes (usingphosphate-buffered saline as vehicle) and boosted three times at 2 weekintervals. Sera were collected 15 days after the last boost and testedin enzyme-linked immunosorbant assay (ELISA) using alkaline phosphataseconjugate-antibodies (Southern Biotechnology) and p-nitrophenylphosphate substrate (Sigma) as the detection system. Cytotoxicity assayswere performed with the splenocytes of the immunized mice: 8×10⁶cells/well were cultured in vitro with B1 peptide (0.4 μM) for 6 days.Effecter cells were tested in a standard 4 h assay using Na₂ ⁵¹ CrO₄(New England Nuclear)-labeled target cells (5×10⁴ cells/well).

RESULTS

The mouse antibody response after i.p. and s.c. immunizations wasevaluated in ELISA as shown in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Induction of Antibody and Cytotoxic Responses                                                 Antibody                                                                              Cytotoxic                                                             Response.sup.a                                                                        Response.sup.b                                                        (titer) (% lysis)                                             ______________________________________                                                        Intraperitoneal Immunization                                  B1M + P3C + liposome.sup.c                                                                      >100      ND.sup.d                                          B1M - P3C         2000      60                                                B1M - P3C/liposome                                                                              7000      80                                                                Subcutaneous Immunization                                     B1M - P3C         700       40                                                B1M - P3C/liposome                                                                              460       98                                                ______________________________________                                         .sup.a Mouse immune sera were tested in ELISA using peptidecoated             microriter plates and alkaline phosphataseconjugate antibody direct           against mouse IgG. The results are expressed as geometric means of the        titers (reciprocal of the serum dilution at which the absorbance produced     by the immune serum is 0.2 units).                                            .sup.b Effecter Tcells were added to syngeneic target cells P815 coated b     the B1 peptide at E:T ratio of 15:1. The values represent the average of      triplicate determinations of a representative experiment.                     .sup.c Control group immunized with B1M not covalently linked to the P3C      lipopeptide and not entrapped in liposomes.                                   .sup.d ND  not done                                                      

The results showed that the induction of anti-V3 antibodies depended onthe route used for the immunization. The i.p inoculation gave 3 to 15fold higher response than s.c. inoculation. Furthermore, the antiserainduced by i.p. immunization were neutralizing. Analysis of the antibodyisotypes in the B1M-P3C/liposome immune sera (FIG. 5) showed that IgG1was by far the dominant subclass. This result is in agreement with otherstudies showing that envelope peptides are almost exclusively IgG1restricted.

The cellular immune response induced by the two preparations was thenanalyzed. The murine splenocytes were restimulated in vitro and testedfor their ability to lyse syngeneic target cells preincubated with theV3 peptide (Table 2). In contrast to a significant difference in thehumoral response induced by the i.p. or s.c. immunization, substantialCTL response was produced by either route of immunization. In addition,a long-term T-cell memory was induced, since there was no decrease inthe cytolytic activity of the immunized mice 5 months after the lastboost with B1M-P3C/liposome. As shown in FIG. 6, the lysis ofpeptide-coated targets paralleled that of gp160-vaccinia infected cells,whereas no killing of untreated targets was observed.

An important requirement for an ideal candidate vaccine is the capacityto induce T-cell responsiveness along with the stimulation of antibodyproduction (31). The above data shows the feasibility to elicit bothhumoral and CTL responses with a peptide-based MAP model (B1M-P3C),without the use of an extraneous adjuvant. The route of i.p. injectionelicits in mice an antibody response stronger than the s.c. inoculation.B1M-P3C in liposomes also provide an useful formulation for presentingthe antigen to the immune system. The rationale for anchoring B1M-P3C onliposomes was to mimic the external appearance of the virion,particularly of the surface protein. Furthermore, the lipid anchor, P3C,serves a dual role, as a built-in adjuvant and a lipid-anchoring moiety.We also show that B1M-P3C induces a long-lasting T-cell immunity. Thisresult is encouraging since it overcomes problems associated withadjuvants such as alum, that do not generate a good cellular response,and with vaccination protocols that do not lead to a lasting T-cellmemory.

DISCUSSION

Described herein is the rational design of a totally syntheticpeptide-based vaccine that induces neutralizing antibody and CTL as wellas a vaccine that is safer and more versatile than a whole virus orviral protein vaccine. As set forth above, a macromolecular assemblageapproach is used to produce a multimeric form of peptide antigen,B1M-P3C, that consists of a lipophilic membrane-anchoring moietycovalently linked to a MAPS core matrix and four dendritic arms ofpeptide antigens derived from the V3 loop of the gp120 envelope proteinof HIV-1. Mice antisera against B1M-P3C neutralize the virus infectivityas shown by the inhibition of syncytium formation and reversetranscriptase, induce T-helper response as shown by the IL-2 production,and elicit CD8+CTL that lyse syngeneic cells expressing gp160 on theircell surface. Furthermore, the B1M-P3C produced long term T-cell memoryas the CTL of the immunized mice remained undiminished 5 months afterthe last boost immunization (24).

These results are particularly pertinent to the development of asynthetic vaccine against AIDS since the predominant vaccine strategyhas focused on neutralizing antibodies rather than cell-mediatedimmunity, which may be an equally effective mechanism to overcomecell-to-cell virus transmission in HIV infection. Subunit proteinadministered with a clinically acceptable adjuvant such as gel ofaluminium or calcium salt is usually insufficient to elicit CTLresponse, particularly CD8 +restricted as shown by the results ofOrentas et al. (25) who found CD4+specific CTL by a gp160 subunitvaccine. Subunit vaccine expressed by live vectors such as vacciniavirus (26) may overcome this limitation but will need furtherdevelopment to define various adverse reactions in humans. The idealvaccine may be eventually derived from the inactivated whole orattenuated virus, but the risk and long latency associated with theinfection have so far limited the enthusiasm for its development.

EXAMPLE 3

In this example, the preparation and testing of an alternativelipophilic moiety is presented. Specifically, the lipophilic moiety wasprepared from palmitoyl lysine (PL), and as reflected by the ensuingresults, a structure comprising PL of alternating chirality wasparticularly effective. The details of the preparation and testing ofthe lipoMAPs based on this lipophilic moiety follow below.

Materials and Methods

Synthesis of MAP-palmitoyl lysine conjugates with symmetrical corematrix (B2SM-Pln, n=1-4).

The B2SM-Pln were manually synthesized by solid phase peptide synthesison Boc Aln OCH₂ -Pam resin (17) (0.10 mmol/g) using a combination of Bocand Fmoc strategy. Removal of the Fmoc group was carried out by 20%piperidine in dimethylformamide. Removal of Boc group was carried out by50% TFA in dichloromethane followed by washing with dichloromethane andneutralization withdiisopropylethylamine/dichloromethane/dimethylformamide (1:9:11).Couplings of amino acids (4 molar equivalents) were carried out with thecoupling reagent HBTU/diisopropylethylamine in dimethylformamide. Thestepwise syntheses were described below.

Palmitoyl lysine. After removal of the Boc group on the resin(Boc-Ala-OCH₂ -Pam-resin), one or more rounds of Fmoc-Lys(Boc) wascoupled sequentially to the alanyl resin. The N.sup.ε -Boc groups on Lyswere then removed and the palmitic acids (6 molar equivalents) werecoupled by symmetrical arthydride method using dicyclohexylcarbodiimide(3 molar equivalents) to form Fmoc-[Lys(Pal)]_(n) -Ala-OCH₂ -Pam-resin.

Ser-Ser linker. The N.sup.α -Fmoc on the Lys(Pal) was then removed. Twoconsecutive rounds of Boc-Ser (Bzl) were coupled by the HBTU method.

Symmetrical MAP core. After the removal of Boc group on Ser(Bzl),Fmoc-Lys(Boc) was coupled. N.sup.α -Fmoc group on Lys was removed andBoc-β-Ala (0.4 mmol/g) was coupled to furnish the first level branching.After removal of Boc in Boc-β-Ala-Lys(Boc), the above steps wererepeated for a second level branching to give a tetravalent MAP and twolevels of symmetrical branching. Note, 0.8 mmol/g of Boc-β-Ala was usedin the second round of branching.

Peptide antigen. The peptide antigen B2 having an amino acid sequence312-329 of the third variable domain of gp120, the envelopeglycoprotein, of HIV-1 strain IIIB, was coupled stepwise using theBoc/HBTU chemistry. The protecting groups for the trifunctional aminoacids: Thr(Bzl), Ser(Bzl), Lys(CIZ), ArgCTos). For each coupling step,1.6 mmol/g of Boc amino acids were used since the resin had beenamplified from 0.1 mmol/g to 0.4 mmol/g.

Cleavage and workup. The resin (0.5 g) was stirred in HF solution (0.3ml of thiocresol, 0.7 ml of p-cresol and 9.0 ml of HF) at 0° C. for 1 hfollowed by extraction with 8M urea in 0.1M Tris-HCl buffer, pH 7.4. Theorganic scavengers were removed by dialysis against 0.1M Tris-HClbuffer, pH 7.4 with decreasing urea concentration to 0M in 24 hours. Allsynthetic MAP-PLs gave satisfactory results of amino acid analyses andmass spectroscopy (FAB or laser desorption).

Synthesis of MAP-P3C with a disulfide linkage

(P3C-Cys-OMe)₂. To an ice-cold solution of tripalmitoyl-S-glycerylcysteine (P3C) (0.91 g, 1 mmol), dimethylcysteine HCl (0.20 g, 0.6 mmol)and triethylamine (1.2 mmol) in THF (5 ml), HOBt (0.14 g, 1 mmol) andDCC (0.23 g, 1.05 mmol) were added. The solution was stirred at 0° C.for 1 hr and at room temperature for 1 hr. Ethyl acetate (10 ml),chloroform (50 ml) and saturated NaHCO₃ were added, and the resultingsolution was then washed with 5% citric acid, NaHCO₃ and water (3 mleach). After being dried over Na₂ SO₄ and concentrated, pure product(0.63 g) was obtained by recrystallization from hexane, m.p., 77.0-78.0C, R_(f) =0.90 (ethylacetate:hexane/3:7).

P3C-Cys-OMe. To a degassed suspension of (P3C-Cys-OMe)₂ (2.46 g, 1.20mmol) in chloroform, triethylamine (0.39 mL0 and DTr (0.82 g, 5.20mmol.) were added under nitrogen. The clear solution was stirred for 2hr, washed with 5% citric acid (3×25 ml) and water (2×25 ml), and driedover Na₂ SO₄. The colorless solid was obtained (1.96 g 80% yield) byrecrystallization from methanol and drying over P₂ O₅ under vacuum, m.p.75°-77° C. R_(f) =0.76 (ethylacetate:hexane/3:7).

P3C-Cys(Pys)-OMe. The solution of P3C-Cys-OMe in chloroform (10 ml) wasadded dropwise into the solution of 2,2'-dithiopyridine (0.95 g) andglacial acetic acid (0.15 ml) in absolute ethanol (4 ml). The mixturewas stirred for 20 hr. During this period, the solution color turnedyellow due to release of 2-pyridinethionol. The solution as concentratedto about 3 ml and passed through a silica gel column(ethylacetate:hexane/1:2 to 2:0). The first colorless fraction was theproduct (1.47 g, 72.3%), m.p. 68.0°-69.0° C., R_(f) =0.78(ethylacetate:hexane/2:l). ¹ HNMR, 350 mHz (FIG. 10). ¹³ CNMR 350 mHz(FIG. 11). Cald: C 66.55, N 3.70, H 10.04, S 8.46. Found: C 66.79, N3.65, H 9.90, S 8.,45, MS: m/z+1137.

B2M-Cys(P3C-Cys-OMe)-Ala. B2M-Cys(Acm)-Ala-Pam-resin was synthesized inFmoc/TFA chemistry as described above. The protecting groupacetamidomethyl (Acm) on the cysteine was removed with Hg(OAc)₂ on theresin as described (12). The B2M-Cys-Ala-Pam resin (10 μmol) inchloroform/propanol (4:1, 8 ml) reacted with P3C-Cys(PyS)-OMe (45 mg, 40μmol) in the presence of triethylamine for 24 hr. The yellow by-product2-pyridinethiol released quantitatively (UV 343 nm, ε=8080), which wasused to monitor the reaction. The unreacted P3C-Cys(PyS)-OMe wasrecovered in the washings with methanol. The yield was 33 to 54%.Satisfactory results of amino acid analyses were obtained afteroxidization of cysteine with performic acid. The product was purifiedthrough gel filtration (Sephadex G75) or directly used for preparationof liposomes, since unreacted MAP peptide did not incorporate intoliposomes and was separated by gel filtration later.

Preparation of liposomes

MAP-PLs were incorporated into liposome with detergent-dialysis method(13). In a round-bottle flask, phosphatidyl choline (5 mg), cholesterol(5 mg) and octylglucoside (100 mg) were dissolved in about 1 ml ofacetone and evaporated under nitrogen. The residue was re-dissolved indiethyl ether and re-evaporated to form a lipid film on the glass wall.A detergent solution (23 mg of octylglucoside in 5 ml of 0.30M NaClaqueous solution) containing 0.5 mg of MAP-PLs was added to the drylipid film and dispersed with vortexing. The resulting clear liquid wasdialyzed for 36 hr against three 2-liter changes of PBS (pH 7.4).Purification of liposome monitored at 225 nm was carried out by gelfiltration on sterile Sephadex G-150 with PBS.

Immunization procedure

Outbred female CD-1 mice or inbred female BALB/c mice (JacksonLaboratories, Bar Harbor, Me.) were injected intraperitoneally with 50μg of the antigen, free or in liposomes, using phosphate-buffered salineas vehicle, for three times at two week intervals. Sera were collectedfifteen days after the last boost. The antibody response was analyzed byenzyme-linked immunosorbent assay (ELISA) using plates coated with B2peptide (5 μ/g well) or gp120 (1 μg/well), kindly provided by Dr. A.Profy (Repligen). Serial dilutions of the sera were added to the wellsand the bound antibodies were detected using goat alkalinephosphatase-conjugate antibody (Sigma) and p-nitrophenyl phosphatesubstrate (Sigma).

Cytotoxicity Assay

Spleen cells from the immunized BALB/c mice were cultured in vitro for 5days in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mMglutamine, 5×10⁻⁵ M 2-mercaptoethanol and antibiotics) in presence of B2peptide (0.4 μM).

The cytotoxic activity was tested in a 4 hr assay against ⁵¹ Cr-labeledsyngeneic P815 cells untreated or preincubated with B2 peptide (0.8 μM).The percentage of specific ⁵¹ Cr release was calculated as 100(experimental release--spontaneous release)/(maximumrelease--spontaneous release). Maximum release was determined fromsupernatants of P815 cells lysed by the addition of 5% Triton X-100 andspontaneous release from target cells incubated alone.

RESULTS

Synthesis of MAP-PLs conjugates

The basic design of the model used for our study in shown in FIG. 1. Itconsisted of four components: an antigen, a core matrix, a hydrophiliclinker and a lipophilic carboxyl end. The selected peptide antigen forthis study was an 18-residue peptide, (amino acid sequence 312-329;referred to as B2), which is derived from the third variable domain (V3loop) of gp120, the envelope glycoprotein of HIV-1 strain IIIB (4). This18-residue sequence includes T-helper and T-cytotoxic epitopes (6) andhas been shown to elicit excellent antibody titers in mice using the MAPsystem in Freund's adjuvant (16), or as a covalent conjugate with theP3C in liposomes (32). The branching core matrix was made by three unitsof β-alanyl-lysine. Since β-alanyl-lysine contains amino groups nearlyequal in distance from the α-carbon of the lysine, the resulting corematrix is nearly symmetrical. It may also have the advantage of greaterflexibility than the conventional asymmetric core matrix consisting onlyof lysine. The carboxyl end of the core matrix contains a hydrophilicdipeptide linker, Ser-Ser, followed by a series of palmitoyl lysineswhich are used as anchors to the lipid matrix. From molecular modelingstudy (Quanta, Silicon Graphic), these palmitoyl lysines are bestpositioned in alternating chirality (D or L) so that the lipid anchorsare in parallel orientation required for inserting into liposomemembrane.

The synthesis of these models was carried out stepwise by thesolid-phase method and was not much different from the methodology thatour laboratory first reported for the preparation of MAP systems. Sincethe conjugations of all parts in these MAP-PLs are amide bonds, they canbe synthesized stepwise as a complete unit by the Merrifield solid phasemethod using a combination of Boc and Fmoc chemistry. The lipophiliccarboxyl peptides could be built by two methods. In the first method,palmitoyl lysine was incorporated as Boc-Lys(Fmoc) and then the Fmocgroup was removed so that palmitic acid was conjugated to the sidechain. Alternatively, the palmitoyl lysine could be premade in solutionchemistry and incorporated as a single unit.

Next, to form the core matrix, β-Ala-Lys was incorporated sequentiallyas Fmoc-Lys(Boc). Deprotection of the Fmoc and incorporation of theBoc-β-Ala produced the desired β-Ala-Lys unit on the resins. Again, apremade dipeptide of Boc-β-Ala-Lys(Boc) could be incorporated as asingle unit to reduce several repetitive steps in solid phasemanipulations. Once the core matrix was completed, the antigens to beamplified fourfold were synthesized sequentially to produce the desiredmodel. The MAP-PLs containing antigens and palmitoyl lysine were cleavedfrom the resin, extensively dialyzed against decreasing concentrationsof urea-buffer solution and then purified by gel permeationchromatography. The products showed the expected amino acid ratio byamino acid analysis. Seven MAP-PLs were prepared to investigate thevarious structural contributions to immunogenicity (FIG. 4).

Immunogenicity of MAP-PLs in liposomes

Since MAP-PLs were developed as a simple replacement of P3C, a lipoMAPmodel containing three palmitoyl lysine substitutions, B2SM-PL3, a MAPcontaining four B2 peptides and three palmitoyl lysines on a symmetricalcore matrix (FIG. 5C) was tested as a prototype. The humoral responseelicited by B2SM-PL3 in mice was analyzed by ELISA. Immunization withB2SM-PL3 in liposomes (B2SM-PL3/lip) elicited antibody response, whileB2SM-PL3 alone was not immunogenic (FIG. 5). Furthermore, the responseof PL3 was specific, since the same construct with palmitic acidsconjugated on the serine side chains such as B2SM-PS3 (FIG. 5A) elicitedsignificantly lower titers than B2SM-PL3 (FIG. 5).

An important question was whether the lipid side chains served solelysame as a depot and required a lipid matrix for its correctpresentation. The immunogenicity of B2SM-PL3 was then compared indifferent aqueous and oil-based formulations. In PBS or alum, nosignificant antibody response was obtained. Only in oil-emulsion or inliposome, did B2SM-PL3 produce significant antibody response (Table 3).

Effect of the linker

A hydrophilic Ser-Ser linker (18) was inserted between the aminoantigen-core matrix and carboxyl lipophilic palmitoyl lysines. Such alinker could exert conformational influence on the overall presentationof our models. To test its conformational influence of MAP-PLs, wecompared the linker L-Ser-L-Ser with L-Ser-D-Ser in the B2SM-PL3 andB2SM-D-PL3 models, respectively (FIG. 4B).

With L-Ser-L-Ser as the linker, we envisioned that this linker wouldallow the antigens to be extruded from the lipid matrix. However,L-Ser-D-Ser imparts a reverse turn to the conjugate and we envisionedsuch a turn might cause the lipid portion containing the palmitoyllysines to fold back to the antigen-core matrix, leading to a completelydifferent presentation of antigens in the liposomal matrix. Indeed,B2SM-PL3 in liposomes was immunogenic, while B2SM-D-PL3 which containedthe L-Ser-D-Ser linker was weakly immunogenic after three immunizationsin mice (FIG. 5).

Effect of numbers of palmitoyl side chain

The immunological responses of B2M-PL3 containing three palmitoyllysines was in general not as good as B2M-P3C containing three palmiticacids on a cysteine.

However, an inherent advantage to the design of our lipoMAP models wasthe flexibility to incorporate different numbers of lipid palmitoyl sidechains as palmitoyl lysines. To investigate the effect of an optimalnumber of lipid side chains on immunogenicity, we prepared five modelsof B2SM containing 0 to 4 palmitoyl lysines, B2SM, B2SM-PL1, B2SM-PL2,B2SM-PL3 and B2SM-PL4 (FIG. 4C).

B2SM-PLs were used to immunize mice alone or incorporated in liposomes.Three of the five models containing 0, 1 and 4 palmitoyl lysines, B2SM,B2SM-PL1 and B2SM-PL4, showed no or very low responses after fourimmunizations. In the presence of liposomes, virtually no incorporationwas observed in the case of B2SM without PL. We found that B2SM-PL1 witha single lipid anchor was very poorly incorporated in the liposomes, andit was very difficult to reach a final concentration of 50 μg of peptidebound to liposomes needed for the immunization. The mice were thereforeimmunized with B2SM-PL2 (two palmitoyl lysines), B2SM-PL3 (threepalmitoyl lysines) and B2SM-PL4 (four palmitoyl lysines) in liposomes oras free constructs, while B2SM (without palmitoyl lysine) and B2SM-PL1(one palmitoyl lysine) were immunized only as free constructs. The bestresponse was obtained from B2SM-PL2/liposomes. As determined by ELISAagainst the gp120 peptide (FIG. 6A) and the native protein (FIG. 6B),B2SM-PL2 elicited higher titers than B2SM-PL3. B2SM-PL4 was notimmunogenic. No antibody response was detected in the mice immunizedwith the same concentrations using the free B2SM-PLs.

Induction of cytotoxic T lymphocytes (CTLs)

We then turned our attention to the induction of CTLs which is animportant contributor to the immunity against vital infections.Recently, several groups have demonstrated in vivo CTLs priming withpeptides or soluble proteins. A common feature in these preparations wasthe presence of lipophilic moieties attached to peptides (14) or the useof hydrophobic adjuvants, such as Freund's adjuvant or ISCOMs (9,33,28).To assess the induction of CTL response after immunization in ourlipoMAP system, spleen cells of the immunized mice were tested for theirability to lyse P815 syngeneic target cells sensitized with B2M peptide.Induction of CTL response, following immunization with the MAP-PLsystem, was analyzed in BALB/c mice (FIG. 7). Strong cytolytic activitywas found in the spleen of the animals immunized with B2SM-PL2 free orin liposomes, indicating that immunodominant cytotoxic epitopes can becoupled to the palmitoyl lysines to raise CTLs in vivo.

Comparison with B2M-P3C

Previously, we have synthesized B2M-P3C, in which P3C was conjugated tothe side chain of lysine at the carboxyl terrains, and found that itelicited both humoral and CTLs when incorporated in liposomes (34-36).However, such synthesis of B2M-PC3 using the linkage to the side chainof lysine lacked flexibility. We therefore developed a modular approach(FIG. 8) to the synthesis of B2M-P3C using the thiol side chain ofcysteine. It was synthesized by conjugating B2M while it was stillattached to the resin to P3C by a disulfide linkage in the solid phase(FIG. 9). The MAP containing a cysteinyl residue at the carboxylterminus and was then conjugated to a Cys-P3C containing a thiopyridineresidue. The advantage of conjugating in the solid phase was that allside products and excess reagents could be removed by washings. Theyield of the reaction was 33-54%. However, those B2SM lacking P3C wereremoved during either the gel permeation chromatography or theincorporation to liposomes. The immunological characteristics of themodular B2M-P3C was compared with those of the new model of B2SM-PL2 andB2SM-PL3. The results are shown in Table 3, below.

                  TABLE 3                                                         ______________________________________                                        Comparison of B2SM - PL3 and B2M - P3C                                               Titers (10.sup.3)                                                             B2SM - PL2                                                                              B2SM - PL3  B2M - P3C                                        ______________________________________                                        PBS      <0.1        0.1         2                                            Alum     ND          <0.1        40                                           Oil emulsion                                                                           ND          20          2.2                                          Liposome 2.0         1.7         1                                            ______________________________________                                    

DISCUSSION

The task of transforming a synthetic peptide antigen into aself-sufficient immunogen capable of eliciting both humoral andcell-mediate responses is challenging. Our results show that the newlipoMAP system with appending, dendritic PLs in a lipid matrix such asliposomes may provide a useful solution. The new system may also be usedto further our understanding of immunogenicity and the roles played bythe adjuvants.

Adjuvants are known to induce nonspecific B or T-cell proliferations bythe induction of cytokines (37). They may also provide a depot for theslow release of the peptide antigens (38). The lipid chains on theIipoMAP appear to provide one or more of these roles. However, itremains to be determined whether the PLs in our lipoMAP play a role toinduce cytokines. Our preliminary results have shown that MAP-PLs arenot B-cell mitogens (data not shown) and differ from P3C in this aspect.

The advantages of a built-in adjuvant on a peptide antigen have beenshown by Chedid and his co-workers (39-41) using derivatives of themuramyl dipeptide, a component of the Freund's adjuvant. Lipid moletieshave been used as covalent attachment to the amino terminus, mainly forthe purpose of increasing the ability of the peptide antigen to serve asa depot (42-43, 48). We have found that adding lipid moiety to theantigens often alters their immunogencity and have approached thisproblem in a systematic manner in the design of the lipoMAP system.First, we examine the role of a more flexible core matrix containing aβ-alanyl lysine as a building unit which will provide less stericcrowding than that using lysine alone. However, the difference of theconventional asymmetric or the new, symmetrical core matrix to improvethe presentation of epitope peptide and hence to enhance the antibodyresponse does not appear to be an important factor. We have found thatthere is essentially no difference in immune response using either corematrix in our model with three PLs (43).

The hydrophilic linker Ser-Ser appears to be important since the use ofthe Ser-D-Ser linker which orients the lipid portions of the compounddifferently did not provide the desired immunogenicity. The role oflipid as depot appears to be important since B2SM-PL3 in alum or PBSalone did not result in any significant immunological response. Onlythose B2SM-PL3 in liposomes (34-35) or in oil-emulsion (36) inducesignificant antibody response.

We next tested the number of lipid side chains. The optimal numberappears to be two. This structural similarity is important for theincorporation and rigid orientation of MAP-PLs on liposomes. The lowresponse of B2SM-PL1 may due to its inability to anchor in liposomeswhile B2SM-PL3 and B2SM-PL4 have lipid tails crossing each other asshown by molecular simulation. The stereospecific requirements of lipidattachments in Lipid A have been clearly shown by Shiba and hiscoworkers who have found that altering the number or chirality of thelipid side chains of Lipid A leads to less potent molecules. Similarly,Jung and his coworkers have shown that the chirality (and theorientation) of the palmitoyl side chains of P3C is important formitogenicity. These results imply that the design of IipoMAP may requireto take into consideration the specific conformation of lipid sidechains in addition to the presenting peptide antigens on the surfaces ofliposomes.

Finally, we have also focused our design on the ability of the peptideantigen to elicit CTLs. We have found that the attachment of PLs with orwithout the aid of liposomes can induce CTLs capable of killingsyngeneic cells expressing gp120 on their cell surfaces (37,45-47).These results show that the processing of B- and T-cell antigens havedifferent requirements. More importantly, it shows the versatility oflipoMAP to elicit both humoral and cell-mediated responses. Thesimplicity in the design of the lipoMAP and its versatility may be auseful tool for many mechanistic investigations.

The following is a listing of certain of the publications referred to inabbreviated fashion in the foregoing specification, with numberscorresponding to those appearing hereinabove.

1. Grey, H. M. & Chesnut, R. (1985) lmmunol. Today 6: 101-106.

2. Townsend, A. R., Rothbard, M. J., Gotch, F. M., Bahadur, G., Wraith,D. & McMichael, A. J. (1986) Cell 44: 959-968.

3. Unanue, E. R. & Allen, P. M. (1987) Science 236: 551-557.

4. Robey, W., Arthur, L., Matthews, T., Langlois, A., Copeland, T.,Lerche, N., Oroszlan, S., Bolognesi, D., Gilden, R. & Fischinger, P.(1986) Proc. Natl. Acad. Sci. USA 83: 7023-7027.

5. Javaherian, K., Langlois, A., McDanal, C., Ross, K., Eckler, L.,Jellis, C., Profy, A., Rusehe, J., Bolognesi, D., Putney, S. & Matthews,T. (1989) Proc. Natl. Acad. Sci. USA 86: 6768-6772.

6. Takahashi, H., Merli, S., Putney, S. D., Houghten, R., Moss, R.,Germain, R. N. & Berzofsky, J. A. (1989) Science 246: 118-121.

7. Lerner, R. A. (1982) Nature 299: 592-596.

8. DiMarchi, R., Brooke, G., Gale, C., Cracknell, V., Doel, T. & Mowat,N. (1986) Science 232: 639-641.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A multiple antigen peptide system comprising adendritic core to which are covalently attached at least one peptide anda lipophilic membrane anchoring moiety, wherein said multiple antigenpeptide system exhibits adjuvant properties and when injected into amammal, is capable of cliciting a full immune response provided by bothhumoral and cell mediated immunities including a cytotoxic T lymphocyteimmune response.
 2. The multiple antigen peptide system of claim 1wherein said lipophilic membrane anchoring moiety comprises aconstituent selected from the group consisting of a lipoamino acid, aliposome, a saponin derivative alone or in admixture with cholesterol,and a suitable surfactant material.
 3. The multiple antigen peptidesystem of claim 2, wherein said lipophilic membrane anchoring moietycomprises a lipoamino acid.
 4. The multiple antigen peptide system ofclaim 1 wherein said dendritic core comprises a bifunctional unit. 5.The multiple antigen peptide system of claim 1 further comprising acovalently attached T cell epitope.
 6. The multiple antigen peptidesystem of claim 3 wherein said lipoamino acid is derived from aminoacids selected from the group consisting of cysteine, lysine, serine andmixtures thereof.
 7. The multiple antigen peptide system of claim 6wherein said lipophilic membrane anchoring moiety comprisestripalmitoyl-S-glycerylcysteine.
 8. The multiple antigen peptide systemof claim 6 wherein said lipophilic membrane anchoring moiety comprisesdipalmitoyl-S-glycerylcysteine.
 9. The multiple antigen peptide systemof claim 6 wherein said lipophilic membrane anchoring moiety comprisespalmitoyl lysine.
 10. The multiple antigen peptide system of claim 3wherein said lipoamino acid is covalently attached through a peptidebond to an amino acid polymer comprising a peptide.
 11. The multipleantigen peptide system of claim 10 wherein said peptide is alipopeptide.
 12. The multiple antigen peptide system of claim 5 whereinsaid T cell epitope is covalently linked to said peptide.
 13. Themultiple antigen peptide system of claim 12 wherein said T cell epitopeis covalently linked in tandem to said peptide.
 14. The multiple antigenpeptide system of claim 5 wherein said T cell epitope is at least sevenamino acids long.
 15. The multiple antigen peptide system of claim 5wherein the T cell epitope is a cytotoxic T cell epitope.
 16. Themultiple antigen peptide system of claim 5 wherein the T cell epitope isa helper T cell epitope.
 17. The multiple antigen peptide system ofclaim 5 wherein the T cell epitope is derived from an HIV-1 protein. 18.The multiple antigen peptide system of claim 17 wherein the HIV-1protein is the HIV-1 envelope glycoprotein.
 19. The multiple antigenpeptide system of claim 1 wherein said system is encapsulated within aliposome.
 20. The multiple antigen peptide system of claim 1 whereinsaid dendritic core comprises lysine.
 21. The multiple antigen peptidesystem of claim 1 wherein said peptide is between 10 and 40 amino acidslong.
 22. The multiple antigen peptide system of claim 5 furthercomprising a B cell epitope.
 23. The multiple antigen peptide system ofclaim 22 wherein the B cell epitope and the T cell epitope are linked onthe same functional group of the dendritic core.
 24. The multipleantigen peptide system of claim 4 wherein said dendritic core istetravalent.
 25. The multiple antigen peptide system of claim 2 whereinsaid suitable surfactant material comprises a mixture of long chainpolyoxyethylenes and polyoxypropylenes.
 26. The multiple antigen peptidesystem of claim 4 wherein the bifunctional unit comprises an amino acidselected from the group consisting of cysteine, lysine, aspartic acid,glutamic acid, and ornithine.
 27. The multiple antigen peptide system ofclaim 26 comprising eight free functional groups in the dendritic coreand eight peptides, wherein each of the eight peptides is attached toeach of the eight free functional groups, thereby forming an octavalentmultiple peptide antigen.
 28. The multiple antigen peptide system ofclaim 27 further comprising eight covalently attached T cell epitopes.29. The multiple antigen peptide system of claim 28 wherein the T cellepitopes are derived from an HIV-1 protein.
 30. The multiple antigenpeptide system of claim 28 wherein the lipophilic membrane anchoringmoiety comprises a constituent selected from the group consisting of alipoamino acid, a liposome, a saponin derivative alone or in admixturewith cholesterol, and a suitable surfactant material.
 31. The multipleantigen peptide system of claim 30 wherein the lipophilic membraneanchoring moiety is a Iipoamino acid derived from an amino acid selectedfrom the group consisting of cysteine, lysine, serine and mixturesthereof.
 32. The multiple antigen peptide system of claim 30 wherein thelipophilic membrane anchoring moiety is a lipoamino acid selected fromthe group consisting of tripahnitoyl-S-glycerylcysteine,dipalmitoyl-S-glycerylcysteine, and palmitoyl lysine.
 33. A method forgenerating antibodies in a mammal, said method comprising administeringto said mammal an antibody-generating amount of the multiple antigenpeptide system of claim
 1. 34. A method for generating antibodies in amammal said method comprising administering to said mammal anantibody-generating amount of the multiple antigen peptide system ofclaim
 32. 35. A method for generating antibodies in a mammal said methodcomprising administering to said mammal an antibody-generating amount ofthe multiple antigen peptide system of claim
 23. 36. A method forgenerating antibodies in a mammal said method comprising administeringto said mammal an antibody-generating amount of the multiple antigenpeptide system of claim 5.